The major objective of this project is to study the role of mitochondria in oxysterol-induced apoptosis of human lymphoblastic leukemia cells. Oxysterols represent a class of potent inducers of apoptosis in various cell types, particularly leukemic lymphoid cells. Oxysterols also act as powerful transcriptional regulators of genes involved in cholesterol biosynthesis, cell growth and apoptosis. Although the molecular basis of oxysterol-induced apoptosis of lymphoid cells remains unknown, some studies have suggested that oxysterol induction of apoptosis is mediated by reactive oxygen species generation. Results obtained in the previous funding cycle show that oxysterol treatment of CEM cells induces a decrease in ATP levels and loss of mitochondrial membrane potential. This project will test the hypothesis that ROS damage particularly to mitochondrial DNA plays a role in oxysterol-induced apoptosis of human leukemia cells. Our hypothesis predicts that oxidative damage to mitochondrial DNA can lead to mitochondrial dysfunction and induction of apoptosis. To test this hypothesis we will: 1) determine whether the formation and repair of oxidative damage to nuclear and mitochondrial DNA are higher in oxysterol sensitive human lymphoid cells than in oxysterol-resistant cells after exposure to oxysterols, 2) determine mitochondrial function and dysfunction in oxysterol-sensitive and resistant human lymphoid cells after exposure to oxysterols, and 3) determine the expression profile of genes involved in oxidative stress, DNA repair and apoptosis during oxysterol-induced apoptosis of lymphoid cells. This project will assist in laying the foundation for a research project directed towards the understanding of the role of mitochondria in the process of apoptosis.